B
IOLOGICALC
ONTROLViability of
Nezara viridula
(L.) Eggs for Parasitism by
Trissolcus
basalis
(Woll.), under Different Storage Techniques in Liquid Nitrogen
Beatriz S. Corrêa-Ferreira1 and Maria C. N. de Oliveira1
1Embrapa Soja, Caixa postal 231, 86001-970, Londrina, PR.
An. Soc. Entomol. Brasil 27(1): 101-107 (1998)
Viabilidade de Ovos de Nezara viridula (L.) ao Parasitismo por Trissolcus basalis (Woll.), sob Diferentes Técnicas de Armazenamento em Nitrogênio Líquido RESUMO - Diferentes técnicas de conservação de ovos foram avaliadas, sob condições de baixa temperatura, visando a multiplicação do parasitóide de ovos de percevejos Trissolcus basalis (Woll.) (Hymenoptera: Scelionidae). Massas de ovos frescos de Nezara viridula (L.) foram armazenadas, por 12 meses, em nitrogênio líquido (-1960C), embaladas em papel alumínio, em tubos de
microcentrífuga (“eppendorf”) e em sacos plásticos duplos vedados a vácuo (500 ovos/técnica/mês). A cada 30 dias, 10 massas de ovos, conservadas sob as diferentes modalidades, foram retiradas, descongeladas imediatamente e submetidas ao parasitismo por T. basalis. Ovos do percevejo verde, estocados a -1960C, apresentaram condições de conservação e viabilidade para o
desenvolvimento de T. basalis, constatando-se elevados índices de parasitismo e emergência nos ovos armazenados sob as três diferentes técnicas. Os ovos conservados em papel alumínio (95,4%), em tubos (92,3%) ou a vácuo (93,7%) apresentaram parasitismo estatísticamente igual aquele verificado em ovos fres-cos (97,3%). A viabilidade dos ovos, expressa pela taxa de emergência dos adultos do parasitóide, indica que as três técnicas estudadas proporcionaram excelentes condições de desenvolvimento e emergência de T. basalis, não havendo diferenças significativas quando comparadas a ovos frescos. Ao longo do período total de armazenamento (12 meses), não houve redução no número de fêmeas geradas por postura, embora tenham ocorrido flutuações na razão sexual, para alguns períodos. Os resultados obtidos viabilizam a conservação dos ovos do percevejo verde, e posterior multiplicação do parasitóide T.basalis, por um período duas vezes mais longo ao que hoje é conhecido e utilizado.
PALAVRAS-CHAVE: Insecta, controle biológico, conservação de ovos de percevejos, baixa temperatura, parasitóide.
immedi-ately and subjected to parasitism by T. basalis. Green stink bug eggs, stored at -196ºC presented conservation conditions and viability for T. basalis develop-ment, with high parasitism rates and emergence in the eggs stored under the three different techniques. Parasitism of eggs stored wrapped in aluminum foil (95.4%), in tubes (92.3%), and under vacuum (93.7%) were statistically equal to parasitism in fresh eggs (97.3%). Egg viability, expressed by the rate of emer-gence of adult parasitoids, indicates that the three techniques studied provided excellent T. basalis development and emergence conditions with no significant differences when compared with fresh eggs. Throughout the total storage pe-riod (12 months) there was no reduction in the number of females hatched, although there were fluctuations in the sexual ratio for some periods. These results make green stink bug egg conservation, and later multiplication of the parasitoid T. basalis possible for a period twice as long as that known and used today.
KEY WORDS: Insect, biological control, stink bug egg conservation, low tem-perature, parasitoid.
In biological control programs which in-volve the release of a great number of para-sites, it is extremely important to preserve the eggs of the hosts for prolonged periods to make better use of the production from a pre-established colony, and to have great quanti-ties of beneficial agents available when field release is necessary.
Egg parasitoids can successfully develop in host eggs that have undergone freezing or heating (Wajnberg & Hassan 1994). Promis-ing results for lepidopterous egg storage for later Trichogramma spp. multiplication and use in biological control studies have been re-ported in India (Dass & Ram 1983) and in China (Huai-Yi 1988, Morrison 1988, Zhenwei & Qiyao 1988).
Eggs from various Heteroptera species can be stored at low temperatures and still be parasited by Scelionidae species (Orr 1988). For stink bug eggs, Powell & Shepard (1982) found that Trissolcus basalis (Woll.) emer-gence was not reduced in Nezara viridula (L.) eggs stored in a freezer and they could be stored successfully for periods up to seven months. Similar results were obtained by Corrêa-Ferreira & Moscardi (1993) with eggs from this stink bug which, when wrapped in aluminum foil, were viable for T. basalis
para-sitism, and by Korneeva et al. (1981), cited by Orr (1988), for storage of Dendrolinus pini (L.) eggs parasited by Telenomus tetratonus Thomson. Popov (1974), cited by Orr (1988), found that the Trissolcus grandis (Thompson) species did not prefer fresh eggs of several Pentatomidae species in relation to eggs storaged at -20°C for more than 270 days, although the researcher found a decrease in the parasitism rate in eggs stored for periods longer than 180 days.
Studies of stink bug egg storage in liquid nitrogen were carried out by Genduzo (1979). This author demonstrated the viability of stor-ing eggs from several Heteroptera species for periods of up to three years and the parasit-ism by Gryon and Trissolcus species were not affected. The usefulness of this method was confirmed by Gennadiev & Khlistovskii, cited by Orr (1988) also for parasitism by Trissolcus simoni (Mayr) in Graphosoma lineatum (L.) eggs stored for five years. M.L. Costa & F.G.V. Schmidt (personal communication), using the same method, obtained viability in N. viridula eggs after six months storage at -196°C with 83.3% parasitism and 79.0% T. basalis adult emergence, however, results for longer conservation periods were not men-tioned.
As the production and conservation of host eggs is still a limiting factor in soybean stink bug biological control programs, different storage techniques in liquid nitrogen were assessed to allow a greater storage period and thus a better use of the N. viridula egg pro-duction for later multiplication and massive release of T. basalis in soybean fields.
Material and Methods
Three storage techniques were compared under laboratory conditions for N. viridula egg viability for parasitism by T. basalis, after dif-ferent storage periods, at low temperatures. The eggs were collected daily from a host in-sect colony, set up in the laboratory follow-ing the methodology described by Corrêa-Ferreira (1985), and kept in liquid nitrogen at -196°C for 12 months, under different types of packaging: eggs wrapped in aluminum foil; eggs placed in microcentrifuge tubes (“eppendorf”), and eggs placed in vacuum sealed double plastic bags.
Approximately 500 non-parasited eggs / technique/month were used to assess the egg storage techniques. Every 30 days, ten egg masses stored under the different methods were taken out and thawed by immediate im-mersion in 30°C water for five seconds, and subjected to parasitism by T. basalis.
Com-parison with parasitism in N. viridula fresh eggs was used as a control. Parasitism of the stored eggs was assessed during 12 months, using glass tubes (8.0 x 2.5 cm) and two-day-old females, previously mated and fed on honey. Each group of ten egg masses was subjected to ten T. basalis females, for 24 hours. After this period, the egg masses were individualized in petri dishes with moist fil-ter paper, and kept in incubators, under con-trolled conditions of temperature (25±2°C), relative humidity (65±10%) and photophase (14h), until emergence and death of the parasitoids. The parasited eggs were later counted and the number of male and females emerged from each egg mass recorded.
The egg viability was assessed monthly for the parasitism rate, adult emergence and sexual ratio. A parasited egg was considered to be any whose adult completed develop-ment. All the adults which emerged, plus those which had some perforation in the parasited egg chorion were used to calculate the emergence percentage. The sexual ratio was calculated by the formula SR= females / males + females. Any egg without emergence was later dissected to confirm its contents.
The experiment layout of the treatments was a factorial completely randomized design, with ten replications. The normality of the errors, model design, constancy of variances for the treatments and error dispersion were assessed for the requirements of the analysis of variance by the W statistic test (Shapiro & Wilk 1965), F of the additivity model (Tukey 1949), Q for the homogeneity of variances (Burr & Foster 1972) and the error test (R.C.P. Parente, unpublished) and the means were compared by the Tukey test to 5% probabil-ity.
Results and Discussion
(> 90%) (Table 1).
Eggs wrapped in aluminum foil had mean parasitism percentages ranging from 89.3% to 99.8%, statistically equal to parasitism re-corded in fresh green stink bug eggs, for every month. Similar results were obtained when the eggs were stored in microcentrifuge tubes,
eggs submited to parasitism in a 12 month storage period (Table 1). These results show that liquid nitrogen storage conditions cor-rected distortions found up to now in previ-ous studies on T. minutum (Lund 1934, Van Stenberg 1934), with T. grandis (Popov 1974 cited by Orr 1988) and with T. basalis
(Corrêa-which had a mean parasitism rate of 92.3%, during a year. Although the results had sig-nificantly different values from the control for some storage periods (3, 6, 7 and 10 months) with the exception of seven months, all the others had parasitism higher than 88% of the eggs offered.
Storing eggs in vacuum sealed double plastic bags also resulted in high mean rates of parasitism, ranging from 86.2% to 99.5%. No reduction was found in egg parasitism for this technique, nor for the others, as storage time at -196°C increased.
The viability of eggs stored at low tem-peratures as expressed by the emergence rate of T. basalis adults showed that the three tested techniques provided ideal conditions for de-velopment and emergence of the parasitoids, and no significant differences were found among the eggs stored at -196°C and fresh
Ferreira & Moscardi 1993), where these au-thors found a decrease in parasitoid emer-gence for periods greater than 180 days stor-age, probably due to the increased desicca-tion of the host eggs. Under nitrogen, stink bug egg storage in aluminum foil, besides being a very practical technique, gave para-sitism (94.2%) and adult emergence (94.6%) higher than 83.3% and 79.0% respectively, obtained by M.L.Costa & F.G.V. Schmidt (personal communication) for storage for six months in double plastic bags at the same tem-peratures.
Throughout the total storage period (12 months) no sharp reduction in the number of females from egg mass was found with in-creased egg storage time for the different tech-niques compared to offspring from fresh eggs (Table 1). Normally the T. basalis offspring , hatching originating in fresh N. viridula eggs, Table 1. Viability of Nezara viridula eggs for parasitism by Trissolcus basalis, under different storage techniques1, during 12 months, in liquid nitrogen (-196ºC).
Viability ( X ± SE)3
Techniques2
Parasitism (%) Emergence (%) Sexual Ratio4
Control 97.3 ± 0.89 98.5 ± 0.41 0.82 ± 0.01
Aluminum 95.4 ± 1.01 97.4 ± 0.71 0.72 ± 0.03
Tube 92.3 ± 2.57 97.9 ± 0.50 0.73 ± 0.04
Vacuum 93.7 ± 1.07 98.2 ± 0.42 0.74 ± 0.02
C. V. (%) 5.51 1.90 12.29
1N= 500 eggs / months / technique.
2Control= fresh eggs; Aluminum= eggs wrapped in aluminum foil; Tube= eggs placed in
microcentrifuge tubes, Vacuum= eggs placed in vacuum sealed double plastic bags.
3No significant differences were found when the different techniques were compared by Tukey
test P< 0.05.
had, in absolute values, mean sex ratios higher than those from eggs stored at low tempera-tures, although real differences were only found for some storage periods and in certain techniques, varying from 0.58 to 0.91 in the 12 month period.
Although packing eggs in aluminum foil has been used under freezer conditions by the T. basalis multiplication laboratories, devel-opment and emergence obtained in liquid ni-trogen in 12 months storage were twice as great as those obtained in the freezer. Throughout the storage time, high mean rates were found which were always greater than 90% for parasitism and emergence, which were not statistically different from the con-trol (Fig 1). There was, however, a greater variation in the sexual ratio of the generation developed in these eggs, with values statisti-cally different from the control for the 5, 6, 8 and 10 month periods. There was no reduc-tion in the proporreduc-tion of females generated with increased storage time, but fluctuations throughout the period, differences which may
be explained by the influence of other factors which are known to affect the sexual ratio of egg parasitoids.
However, there have been advances in Brazil in N. viridula egg conservation meth-odology, with viability for multiplication of the T. basalis parasitoid, the recommended storage methodology of the biological con-trol program (Corrêa-Ferreira 1993) did not allowed the use of the egg production for a greater storage time than six months, whether in a freezer (Corrêa-Ferreira & Moscard 1993) or in liquid nitrogen (M.L. Costa & F.G.V. Schmidt, personal communication).The lack of alternative for egg storage has hindered the use of the stink bug egg production in periods when these insects are most abundant in the field, at the end of the soybean harvest (March-April). This use can now be viabilized with the results obtained in liquid nitrogen for 12 months, providing egg con-servation for double the period, and the pos-sibility of releasing a greater quantity of T. basalis in soybean fields in the following year.
Acknowledgments
We are grateful to Dr. Clara B. Hoffmann-Campo and Dr. Lenita J. de Oliveira (Embra-pa Soja) for critical reading the manuscript and to Joacir de Azevedo for helping in the laboratory work. This is a contribution of Embrapa Soja number 021/97, published with the approval of the technical director
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